Method for stabilizing DNA aptamers

ABSTRACT

An object of the present invention is to provide a convenient and low-cost method for enhancing the stability of a DNA aptamer and/or its capacity to bind to a target molecule, and a DNA aptamer obtained by the method. 
     The object is solved by substituting an internal hairpin structure (stem-loop structure) of the DNA aptamer with a structure called mini-hairpin structure and optionally increasing GC pairs in a stem portion of the DNA aptamer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage application of PCT/JP2016/056805,filed Mar. 4, 2016, which claims priority from Japanese application JP2015-045266, filed Mar. 6, 2015.

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-WEB and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 13, 2017, isnamed sequence.txt and is 8 KB.

TECHNICAL FIELD

The present mention relates to a method for designing a nucleotidesequence that enhances the capacity of a DNA aptamer to hind to a targetmolecule and/or stabilizes the DNA aptamer, and a DNA aptamer havingsuch properties.

BACKGROUND ART

Functional nucleic acids, such as siRNAs, nucleic acid aptamers, anddecoy nucleic acids, have drawn attention as pharmaceuticals ordiagnostic agents in recent years, and research on and development ofvarious nucleic acid drugs and the like are ongoing with the goal ofestablishing medical applications thereof all over the world.

The general problem of nucleic acids, however, is that these nucleicacids are likely to be degraded by nucleolytic enzymes, such asnucleases, in vivo. In vivo stabilization of the nucleic acids isessential problem for nucleic acid drugs to exert the pharmacologicaleffects efficiently and continuously.

For improving the stability of nucleic acid aptamers against nucleolyticenzymes, a general approach involves chemically modifying sugar orphosphate portions, which are the backbones of nucleic acid, molecules(Non Patent Literatures 1 and 2 and Patent Literature 1). The problemsof these modifications, however, are that the modifications might alsoinfluence the higher-order structure or physical properties of thenucleic acids; and the modifications might not only cause reduction inthe capacity to bind to a target molecule or higher in vivo toxicity butincrease production cost. Accordingly, under the present circumstances,it requires to modify modifiable sites, exhaustively screen them, andindividually analyze them in order to practically use chemicallymodified nucleic acid aptamers as pharmaceuticals. Also, it is generallydifficult to improve the capacity to bind to a target molecule by suchchemical modifications, and there have been few reports thereon.

Thus, a convenient and low-cost method for enhancing the stability of anucleic acid aptamer and further improving its capacity to bind to atarget molecule has been demanded.

CITATION LIST Patent Literature

-   Patent Literature 1: European Patent Application Publication No.    1931694

Non Patent Literature

-   Non Patent Literature 1: Peng, C. G., Masad, J., Damha, M. J.,    G-quadruplex induced stabilization by    2′-deoxy-2′-fluoro-D-arabinonucleic acids (2′F-ANA), 2007, Nucl.    Acids Res., 35, pp. 4977-4988.-   Non Patent Literature 2: Wang, R. E., Wu, H., Niu, Y., Cai, J.,    Improving the stability of aptamers by chemical modification, 2011,    Curr. Med. Chem., pp. 4126-4138.

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to provide a convenient andlow-cost method for enhancing the stability of a DNA aptamer and/or itscapacity to bind to a target molecule, and a DNA aptamer obtained by themethod.

Solution to Problem

The present inventors have completed the present invention by findingthat the stability of an existing DNA aptamer and/or its capacity tobind to a target molecule can be enhanced by substituting an internalhairpin structure of the DNA aptamer with a structure calledmini-hairpin structure and further optionally substituting A-T basepairs in a stem structure of the DNA, aptamer with G-C base pairs.

Thus, the present invention encompasses the following aspects:

(1) A method for designing a base sequence that enhances the capacity ofan existing DNA aptamer to bind to a target molecule and/or stabilizesthe aptamer, comprising substituting a hairpin structure consisting ofone pair of stem structure and loop structure of the DNA aptamercomprising at least one stem structure and at least one loop structurewith a mini-hairpin structure, wherein the mini-hairpin structurecomprises: nucleic acid regions (A) to (C) below sequentially ligatedfrom the 5′ end toward the 3′ end: (A) a first nucleic acid regioncomprising 2 to 5 arbitrary nucleotides; (B) a second nucleic acidregion comprising a “gna” or “gnna” base sequence wherein each “n”independently represents either “g”, “t”, “a”, or “c”, a base analogue,or a modified base; and (C) a third nucleic acid region comprising abase sequence complementary to the first nucleic acid region, whereinthe first nucleic acid region and the third nucleic acid region form astem portion by base pairing with each other and the second nucleic acidregion forms a loop portion.

(2) The method according to the above (1), wherein the DNA aptamercomprises at least one base analogue and/or modified base.

(3) The method according to the above (2), wherein the base analogue is7-(2-thienyl)-3H-imidazo[4,5-b]pyridin-3-yl.

(4) The method according to any one of the above (1) to (3), comprisingincreasing one or more GC pails in stem structure other than thatconstituting the mini-hairpin.

(5) The method according to any one of the above (1) to (4), comprisingincreasing one to five GC pairs in the end of the DNA aptamer, when oneof the at least one stem structure constitutes the end of the DNAaptamer.

(6) The method according to any one of the above (1) to (5), comprisingadding the mini-hairpin structure defined in claim 1 to one end of theDNA aptamer.

(7) A method for producing a DNA aptamer being stabilized and/or havingenhanced capacity to bind to a target molecule, comprising the steps of:designing a base sequence of a DNA aptamer in accordance with the methodaccording to any one of the above (1) to (6); and producing a DNAaptamer on the basis of the designed base sequence.

(8) A DNA aptamer being stabilized and/or having enhanced capacity tobind to a target molecule, obtained by a production method comprisingthe steps of designing a base sequence of a DNA aptamer and producing aDNA aptamer on the basis of the designed base sequence, wherein the stepof designing comprises substituting a hairpin structure consisting ofone pair of stem structure and loop structure of an existing DNA aptamercomprising at least one stem structure and at least one loop structurewith a mini-hairpin structure, and wherein the mini-hairpin structurecomprises: nucleic acid regions (A) to (C) below sequentially ligatedfrom the 5′ end toward the 3′ end: (A) a first nucleic acid regioncomprising 2 to 5 arbitrary nucleotides; (B) a second nucleic acidregion comprising a “gna” or “gnna” base sequence wherein each “n”independently represents either “g”, “t”, “a”, “c”, a base analogue, ora modified base; and (C) a third nucleic acid region comprising a basesequence complementary to the first nucleic acid region, wherein thefirst nucleic acid region and the third nucleic acid region form a stemportion by base pairing with each other and the second nucleic acidregion forms a loop portion.

(9) The DNA aptamer according to the above (8), comprising at least onebase analogue and/or modified base.

(10) The DNA aptamer according to the above (9), wherein the baseanalogue is 7-(2-thienyl)-3H-imidazo[4,5-b]pyridin-3-yl.

(11) The DNA aptamer according to any one of the above (8) to (10),wherein the step of designing comprising increasing one or more GC pairsin stem structure other than that constituting the mini-hairpin.

(12) The DNA aptamer according to any one of the above (8) to (11),wherein the step of designing comprises increasing one to five GC pairsin the end of the DNA aptamer, when one of the at least one stemstructure constitutes the end of the DNA aptamer.

(13) The DNA aptamer according to any one of the above (8) to (12),wherein the step of designing comprises adding the mini-hairpinstructure defined in the above (8), to one end of the DNA aptamer.

(14) A DNA aptamer comprising at least one stem structure and at leastone loop structure, wherein at least one hairpin structure located in aregion other than the end of the DNA aptamer and consisting of one pairof stem structure and loop structure comprises: nucleic acid regions (A)to (C) below sequentially ligated from the 5′ end toward the 3′ end: (A)a first nucleic acid region comprising 2 to 5 arbitrary nucleotides; (B)a second nucleic acid region comprising a “gna” or “gnna” base sequencewherein each “n” independently represents either “g”, “t”, “a”, or “c”,a base analogue, or a modified base; and (C) a third nucleic acid regioncomprising a base sequence complementary to the first nucleic acidregion, wherein the first nucleic acid region and the third nucleic acidregion form a stem portion by base pairing with each other and thesecond nucleic acid region forms a loop portion.

(15) The DNA apt-tinier according to the above (14), wherein the totalGC content in the at least one stem structure is at least 75%.

(16) The DNA aptamer according the above (14) or (15), furthercomprising the hairpin structure defined in the above (14) at one end.

(17) A DNA aptamer for IFN-γ consisting of the nucleotide sequence asshown in any of SEQ ID NOs: 6 and 8 to 11.

(18) A DNA aptamer for VEGF consisting of the nucleotide sequence asshown in any of SEQ ID NOs: 19 to 22.

(19) A DNA aptamer for vWF consisting of the nucleotide sequence asshown in any of SEQ ID NOs: 14 to 16.

(20) A pharmaceutical composition comprising the DNA aptamer accordingto any one of the above (8) to (19).

The present invention encompasses the disclosure of JP Patentapplication No. 2015-045266, to which the present application claimspriority.

According to the method of the present invention, a low-cost andconvenient preparation method can stabilize a DNA aptamer, allow the DNAaptamer to secure high stability in vivo, and/or enhance its capacity tobind to a target molecule. As a result, the DNA aptamer can continuouslyexert its pharmacological effect over a long period of time.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows one example of the step 1 of the present invention. FIG.1(A) shows an example of substituting a hairpin structure with amini-hairpin structure. FIG. 1(B) shows an example of substituting ahairpin structure comprising one bulge structure in the stem structurewith a mini-hairpin structure. FIG. 1(C) shows an example ofsubstituting a hairpin structure comprising one internal loop structurein the stem structure with a mini-hairpin structure. In the diagram,reference numeral 101 denotes a stem structure, reference numeral 102denotes a loop structure, reference numeral 103 denotes a hairpinstructure, reference numeral 104 denotes a first nucleic acid region,reference numeral 105 denotes a second nucleic acid region, referencenumeral 106 denotes a third nucleic acid region, reference numeral 107denotes a mini-hairpin structure, reference numeral 108 denotes a bulgestructure, and reference numeral 109 denotes an internal loop structure.

FIG. 2 shows one example of the step 2 of the present invention. FIG.2(A) shows an example of adding the same number of bases as that ofbases constituting a bulge structure to the other strand. FIG. 2(B)shows an example of adding a smaller number of bases than that of basesconstituting a bulge structure to the other strand. In the diagram,reference character S denotes G or C, and reference character S′ denotesa complementary base of S.

FIG. 3 shows one example of the step 2 of the present invention, whereinA or T in a bulge structure is substituted with G or C, and then, acomplementary base of the substituted base is added. In the diagram,reference character W denotes A or T, reference character S denotes G orC, and reference character S′ denotes a complementary base of S.

FIG. 4 shows one example of the step 2 of the present invention. FIG.4(A) shows an example of substituting all of bases constituting aninternal loop with GC pairs. FIG. 4(B) shows an example of substitutinga portion of the bases with GC pairs, wherein this substitution occursat the end of the internal loop structure. FIG. 4(C) shows an example ofsubstituting a portion of the bases with GC pairs, wherein thissubstitution occurs within the internal loop structure. In the diagram,reference character N denotes A, T, G, or C, reference character Sdenotes G or C, and reference character S′ denotes a complementary baseof S.

FIG. 5 shows one example of the step 2 of the present invention. Thisdiagram shows that a bulge structure is finally formed in one strand, asthe same numbers of substitutions with GC pairs are increased in twostrands, when the number of nucleotides constituting an internal loopstructure of one strand differs from that of another strand. In thediagram, reference character N denotes A, T, G, or C, referencecharacter S denotes G or C, and reference character S′ denotes acomplementary base of S.

FIG. 6 is a diagram showing the sequence and secondary structure of eachaptamer for IFN-γ prepared in Examples. The position of an artificialbase Ds is circled. The site at which hairpin in the original Aptamer 49was substituted with mini-hairpin and the site to which mini-hairpin wasadded are boxed with an arrowhead. The site at which an A-T base pairwas substituted with a G-C base pair is boxed.

FIG. 7 is a diagram showing results of testing the competition of eachaptamer for IFN-γ with Aptamer 49. The upper column shows each aptameradded together with P-labeled Aptamer 49 and the presence or absence ofaddition of IFN-γ (−: not added; +: added). The middle column shows thebands of a complex of P-labeled Aptamer 49 and IFN-γ and free P-labeledAptamer 49. The lower column shows the gel shift rate, the inhibitionrate of binding of Aptamer 49 to IFN-γ by each aptamer calculated fromthe gel shift rate, and the relative binding rate calculated bycomparison with the competitive inhibitory capacity of Aptamer 49itself.

FIG. 8 is a diagram showing results of measuring the binding of eachaptamer for IFN-γ to human IFN-γ by surface plasmon resonance. Theabscissa shows time (sec). The ordinate shows resonance unit (RU). Thenumeric value in the diagram denotes a calculated Kd value.

FIG. 9 is a diagram showing results of confirming the stability of eachaptamer for IFN-γ in human serum by gel electrophoresis. The position ofthe band of an undegraded aptamer is indicated by a black triangle.Residual DNA at each time was calculated in percentage from the band ofan undegraded aptamer electrophoresed after 0 hours, 1 hour, 6 hours, 24hours, 48 hours, and 72 hours.

FIG. 10 is a diagram showing results of measuring the Tm value of eachaptamer for IFN-γ. In FIG. 10(A), the abscissa shows temperature, andthe ordinate shows normalized absorbance. In FIG. 10(B), the abscissashows temperature, and the ordinate shows a first derivative of thenormalized absorbance.

FIG. 11 is a diagram showing results of measuring the inhibitory effectof each aptamer for IFN-γ on response to IFN-γ stimulation by FACS usingcultured cells. FIG. 11(A) is a schematic diagram showing that STAT1 isphosphorylated by IFN-γ stimulation. FIG. 11(B) shows results of FACSanalysis using a phosphorylated STAT antibody. The abscissa showsfluorescence intensity. The ordinate shows the number of cells.Unstimulated cells are indicated by an area filled with gray.Unstimulated cells that were not treated with an anti-phosphorylatedSTAT-1 antibody solution are indicated by a line. FIG. 11(C) shows FACSanalysis on IFN-γ treated cells using a phosphorylated STAT antibody.The abscissa shows fluorescence intensity. The ordinate shows the numberof cells. Unstimulated cells are indicated by an area filled with gray.Cells stimulated with IFN-γ are indicated by a line.

FIG. 12 is a diagram showing results of measuring the inhibitory effectof each aptamer for IFN-γ on response to IFN-γ stimulation by FACS usingcultured cells. The abscissa shows fluorescence intensity. The ordinateshows the number of cells. The upper panels show the inhibitory effectof Aptamer 49. The lower panels show the inhibitory effect of Aptamer58.

FIG. 13 is a diagram showing results of measuring the inhibitory effectof each aptamer for IFN-γ on response to IFN-γ stimulation by FACS usingcultured cells. The abscissa shows fluorescence intensity. The ordinateshows the number of cells.

FIG. 14 is a diagram showing the sequence and secondary structure ofeach aptamer for IFN-γ consisting of natural bases, prepared inExamples. The position of an artificial base Ds is circled. The site atwhich hairpin in the original Aptamer 49 was substituted withmini-hairpin and the site to which mini-hairpin was added are boxed withan arrowhead. The site at which an artificial base Ds was substitutedwith A and the site at which an A-T base pair was substituted with a G-Cbase pair are indicated by small boxes.

FIG. 15 is a diagram showing results of testing competition with Aptamer49. The upper column shows each aptamer added together with P-labeledAptamer 49 and the presence or absence of addition of IFN-γ (−: notadded; +: added). The middle column shows the bands of a complex ofP-labeled Aptamer 49 and IFN-γ and free P-labeled Aptamer 49. The lowercolumn shows the gel shift rate, the inhibition rate of binding ofAptamer 49 to IFN-γ by each aptamer calculated from the gel shift rate,and the relative binding rate calculated by comparison with thecompetitive inhibitory capacity of Aptamer 49 itself.

FIG. 16 is a diagram showing results of measuring the Tm value of eachaptamer for IFN-γ consisting of natural bases. In the graph of FIG.16(A), the abscissa shows temperature, and the ordinate shows normalizedabsorbance. In the graph of FIG. 16(B), the abscissa shows temperature,and the ordinate shows a first derivative of the normalized absorbance.

FIG. 17 is a diagram showing the sequence and secondary structure ofeach aptamer for vWF A1 domain prepared in Examples. The site at whichhairpin in the original ARC1172(wt) was substituted with mini-hairpinand the site to which mini-hairpin was added are boxed with anarrowhead. The position of inverted dT added to the end is circled.

FIG. 18 is a diagram showing results of testing the competition of eachaptamer for vWF A1 domain with ARC1172(wt). The upper column shows eachaptamer added together with P-labeled ARC1172(wt) and the presence orabsence of addition of vWF A1 (−: not added; +: added). The middlecolumn shows the bands of a complex of P-labeled ARC1172(wt) and vWF A1and free P-labeled ARC1172(wt). The lower column shows the gel shiftrate, the inhibition rate of binding of ARC1172(wt) to vWF A1 domain byeach aptamer calculated from the gel shift rate, and the relativebinding rate calculated by comparison with the competitive inhibitorycapacity of ARC1172(wt) itself.

FIG. 19 is a diagram showing results of confirming the stability of eachaptamer for vWF A1 domain in human serum by gel electrophoresis. Theposition of the band of an undegraded aptamer is indicated by a blacktriangle. Residual DNA at each time was calculated in percentage fromthe band of an undegraded aptamer electrophoresed after 0 hours, 1 hour,6 hours, 24 hours, 48 hours, and 72 hours.

FIG. 20 is a diagram showing results of measuring the Tm value of eachaptamer for vWF A1 domain. In the graph of FIG. 20(A), the abscissashows temperature, and the ordinate shows normalized absorbance. In thegraph of FIG. 20(B), the abscissa shows temperature, and the ordinateshows a first derivative of the normalized absorbance.

FIG. 21 shows the sequence and secondary structure of each aptamer forVEGF165 prepared in Examples. The position of an artificial base Ds iscircled. The site at which a G-C base pair was added to the originalAptamer 47 and the site at which an A-T base pair was substituted with aG-C base pair are indicated by small boxes. A mini-hairpin sequence isboxed with an arrowhead.

FIG. 22 is a diagram showing results of testing the competition of eachaptamer for VEGF165 with Aptamer 47. The upper column shows each aptameradded together with P-labeled Aptamer 47 and the presence or absence ofaddition of VEGF165 (−: not added; +: added). The middle column showsthe bands of a complex of P-labeled Aptamer 47 and VEGF165 and freeP-labeled Aptamer 47. The lower column shows the gel shift rate, theinhibition rate of binding of Aptamer 47 to VEGF165 by each aptamercalculated from the gel shift rate, and the relative binding ratecalculated by comparison with the competitive inhibitory capacity ofAptamer 47 itself.

FIG. 23 is a diagram showing results of confirming the stability of eachaptamer for VEGF165 in human serum by gel electrophoresis. The positionof the band of an undegraded aptamer is indicated by a black triangle.Residual DNA at each time was calculated in percentage from the hand ofan undegraded aptamer electrophoresed after 0 hours, 1 hours, 24 hours,48 hours, and 72 hours.

FIG. 24 is a diagram showing results of measuring the Tm value of eachaptamer for VEGF165. In the graph of FIG. 24(A), the abscissa showstemperature, and the ordinate shows normalized absorbance. In the graphof FIG. 24(B), the abscissa shows temperature, and the ordinate shows afirst derivative of the normalized absorbance. The graph of FIG. 24(C)is enlargement of a portion up to 50° C. of the graph of FIG. 24(A).

DESCRIPTION OF EMBODIMENTS 1. Definition

The general terms used in the present specification are defined asfollows:

In the present specification, the “nucleic acid” or the “nucleic acidmolecule” refers to a biological polymer that is constituted bynucleotide units linked through phosphodiester bonds, as a rule.

In the present specification, the “natural nucleotide” refers to anaturally occurring nucleotide. Examples thereof include DNAs composedof deoxyribonucleotides having any of the natural bases adenine,guanine, cytosine, and thymine, RNAs composed of ribonucleotides havingany of the natural bases adenine, guanine, cytosine, and uracil, andcombinations thereof. A nucleic acid (molecule) constituted only bynatural nucleotides is referred to as a natural nucleic acid (molecule)in the present specification.

In the present specification, the “non-natural nucleotide” refers to anon-naturally occurring nucleotide constituted by an artificial base. Aphosphate group and a sugar constituting the non-natural nucleotideaccording to the present invention are structurally identical to thoseof the natural nucleotide.

In the present specification, the “base analogue” or the “artificialbase” refers to an artificially constructed chemical substance havingproperties similar to those of the natural base constituting the naturalnucleotide and can form artificial base pairing with its partner baseanalogue (hereinafter, referred to as a “complementary artificial base”the present specification), as in the natural base. In the presentspecification, the “artificial base pairing” refers to base pairingformed between a pair of complementary artificial bases, as in a pair ofcomplementary natural bases adenine and thymine, adenine and uracil, orguanine and cytosine. The artificial base pairing includes a chemicalbond via a hydrogen bond found in the base pairing between naturalbases, a physical bond via the molecular structure-based associationbetween artificial bases, and stacking effects is hydrophobicinteraction.

The “properties similar to those of the natural base” possessed by theartificial base include properties that permit nucleic acid replicationor transcription (including reverse transcription) through thecomplementary of artificial base pairing. The artificial base hasexclusive selectivity in artificial base pairing, as in the naturalbase. Thus, even a nucleic acid molecule comprising a non-naturalnucleotide, as with the natural nucleotide, can be replicated ortranscribed accurately through the complementary between artificialbases, if non-natural nucleotides respectively having a pair ofcomplementary artificial bases are present among substrate nucleotides.This allows, for example, a DNA molecule to be amplified by a nucleicacid amplification method such as PCR, while the molecule comprises anon-natural nucleotide.

Specific examples of the artificial base include Ds(7-(2-thienyl)imidazo[4,5-b]pyridine; referred to as “Ds” in the presentspecification), Pn (2-nitropyrrole-1-yl; referred to as “Pn” in thepresent specification), Pa (2-formyl-1H-pyrrole-1-yl; referred to as“Pa” in the present specification), P(2-amino-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one; referred to as “P” inthe present specification), Z (6-amino-5-nitro-2(1H)-pyridone; referredto as “Z” in the present specification), 5SICS(6-methylisoquinoline-1(2H)-thione; referred to as “5SICS” in thepresent specification), NaM (3-methoxynaphthalen-2-yl; referred to as“NaM” in the present specification), and MMO2 (2-methoxy-4-methylphenyl;referred to as “MMO2” in the present specification). Examples of thecomplementary artificial base of the artificial base Ds include Pn andPa. Examples of the complementary artificial base of P include Z.Examples of the complementary artificial base of 5SICS include NaM andMMO2.

In the absence of a non-natural nucleotide having a complementaryartificial base in substrates, the artificial base can instead pair witha natural base similar in structure and/or properties to thecomplementary artificial base during replication or transcription. Inthis case, the non-natural nucleotide in the templated nucleic acidmolecule is replaced with a natural nucleotide after replication ortranscription. For example, Ds is known to be replaced with A or T.

In the present specification, the “modified base” means an artificiallychemically modified base. Examples of the modified base include modifiedpyrimidine (e.g., 5-hydroxycytosine, 5-fluorouracil, 4-thiouracil,5-(3-indole-2-ethyl)uracil, and 5-(4-hydroxyphenyl-2-ethyl)uracil),modified purine (e.g., 6-methyladenine and 6-thioguanosine), and otherheterocyclic bases.

In the present specification, the “nucleic acid aptamer” refers to anaptamer constituted by a nucleic acid and refers to a ligand moleculethat is able to strongly and specifically bind to a target moleculethrough the secondary structure of a single-stranded nucleic acidmolecule and further the conformation formed on the basis of a tertiarystructure via a hydrogen bond or the like, thereby specificallyinhibiting or suppressing the functions (e.g., biological activity) ofthe target molecule. Thus, the nucleic acid aptamer can serve as aninhibitor of a target molecule function. In the present specification,the “functional inhibition of a target molecule” refers to inhibition orsuppression of the catalytic function or gene expression controlfunction (including control of transcription, translation, transport,etc.) and/or biological function such as apoptosis control function ofthe target molecule.

The nucleic acid aptamer is generally known as RNA aptamers consistingof RNAs and DNA aptamers consisting of DNAs. In the presentspecification, nucleic acids constituting a nucleic acid aptamer areDNA.

In the present specification, the “target molecule” refers to asubstance that can serve as a target to which the DNA aptamer binds. Thetype of the target molecule is not particularly limited as long as thetarget molecule is a biomaterial to which the DNA aptamer can bind.Examples thereof include peptides (oligopeptides and polypeptides),nucleic acids, lipids, sugars (including sugar chains), andlow-molecular-weight compounds. The target molecule is preferably apeptide, more preferably a polypeptide, i.e., a protein. Alternatively,the target molecule may be any of naturally derived substances,chemically synthesized substances, recombinant substances, cells,viruses, and the like.

2. Method for Designing DNA Aptamer

According to the first embodiment, the present invention relates to amethod for designing a nucleotide sequence that enhances the capacity ofan existing DNA aptamer to bind to a target molecule and/or stabilizesthe aptamer. The present invention particularly relates to a methodcapable of enhancing the capacity of a DNA aptamer to bind to a targetmolecule and/or stabilizing the aptamer on the basis of information atthe secondary structure level of the DNA aptamer. The method fordesigning a nucleotide sequence according to the present inventioncomprises step 1 as an essential step. Also, the method for designing anucleotide sequence according to the present invention comprises one ormore of step 2, step 3, and step 4 as optional steps. When the method ofthe present in comprises these optional steps, the order of the steps isnot limited. However, when the method of the present invention comprisesboth of step 3 and step 4, the step 4 is carried out after the step 3.Each step constituting the method for designing a nucleotide sequenceaccording to the present invention will be described below.

2-1. Step 1

In the present specification, the “step 1” is the step of substituting ahairpin structure consisting of one pair of stem structure and loopstructure of the existing DNA aptamer comprising at least one stemstructure and at least one loop structure with a mini-hairpin structure.

In the present specification, the “stem structure” means adouble-stranded structure formed by the complete or partial base pairingof a portion of bases, preferably 2 or more consecutive, for example 3or more, 4 or more, or 5 or more consecutive bases constituting onestrand with bases constituting another strand. In the presentspecification, the “complete” base pairing means that all of 2 or moreconsecutive, for example, 3 or more, 4 or more, or 5 or more consecutivebases in one nucleotide sequence of a DNA aptamer are base-paired withthe corresponding bases of another nucleotide sequence. The “partial”base pairing means that 1 or 2 or more, for example, 3 or more 4 ormore, or 5 or more unpaired bases are contained in the completelybase-paired nucleotide sequences of the stem structure. Thus, in thiscase, at least one internal loop structure and/or at least one bulgestructure is formed within the stem structure. In the presentspecification, the “internal loop structure” refers to a loop structurethat is formed within the stem structure, when at least one unpairedbase is present at each of the corresponding positions of two strandsconstituting the stem structure. In the present specification, the“bulge structure” refers to a protruding structure that is formed withinthe stem structure, when at least one unpaired base is present at eitherof the corresponding positions of two strands constituting the stemstructure.

In the present specification, the “loop structure” means an unpairedloop-shaped structure that is positioned in two strands constituting thestem structure and formed within the nucleic acid by the formation ofthe stem structure.

In the present specification, the “hairpin structure” or the “stem-loopstructure” means a structure consisting of one stem structure and oneloop structure (one pair of stem structure and loop structure).

In the present specification, the “mini-hairpin structure” has astructure where three DNA nucleic acid regions, i.e., a first nucleicacid region, a second nucleic acid region, and a third nucleic acidregion, are sequentially ligated from the 5′ end toward the 3′ end.

The “first nucleic acid region” is a nucleic acid region comprising 2 to5 arbitrary nucleotides. The nucleotides refer to deoxyribonucleotideshaving a base guanine (g), adenine (a), cytosine (c), or thymine (t).The base of this nucleic acid region is preferably guanine or cytosine.This is because, for forming a stem structure with the third nucleicacid region mentioned later, a larger gc content elevates a Tm value andallows the stem structure to be stably retained. Thus, the wholenucleotide sequence of the first nucleic acid region most preferablyconsists of g and/or c.

The “second nucleic acid region” is a nucleic acid region comprising a5′-gna-3′ or 5′-gnna-3′ nucleotide sequence. In the sequence, each nindependently represents a natural base (g, a, t, or c), the baseanalogue, or the modified base.

The “third nucleic acid region” is a nucleic acid region having anucleotide sequence complementary to that of the first nucleic acidregion. Thus, the nucleotide sequence of the third nucleic acid regionis determined by the nucleotide sequence of the first nucleic acidregion. Also, the first nucleic acid region and the third nucleic acidregion are based-paired with each other within the molecule. As aresult, the first nucleic acid region and the third nucleic acid regionform a stem portion by complete base pairing with each other, and thesecond nucleic acid region between the first nucleic acid region and thethird nucleic acid region forms a loop portion. For example, 7 to 14nucleotide mini-hairpin DNA having the nucleotide sequence of SEQ ID NO:1, 2, 23, or 24 is formed as a whole.

A DNA aptamer of interest of the step 1 is an existing DNA aptamercomprising at least one stem structure and at least one loop structure.In the present specification, the “existing DNA aptamer” is a known DNAaptamer or a DNA aptamer obtained by a method known in the art whosenucleotide sequence has been revealed or can be revealed. In the case ofpreparing a DNA aptamer, the DNA aptamer can be prepared by, forexample, in vitro selection using a modified version of known SELEX(systematic evolution of ligands by exponential enrichment). The SELEXmethod involves selecting nucleic acid molecules bound to a targetmolecule from an pool composed of nucleic acid molecules having randomsequence regions and primer-binding regions at both ends thereof,amplifying the nucleic acid molecules after recovery, and then using themolecules as a nucleic acid pool for the subsequent round. This seriesof cycles is repeated at several to several tens of rounds to selectnucleic acid(s) having higher binding strength against the targetmolecule. The modified version of SELEX involves, in addition to thesesteps of the conventional SELEX method, the step of immobilizing acomplex obtained by the mixing of a nucleic acid pool with a targetmolecule onto a solid-phase carrier. For the details of the modifiedversion of SELEX, see WO2013/073602. The DNA molecule finally obtainedby such a method can be used as the DNA aptamer.

Those skilled in the art can readily identify a nucleotide sequenceconstituting the stem structure and the loop structure in the DNAaptamer by predicting a secondary structure on the basis of thenucleotide sequence thereof. For the prediction of a secondary structureon the basis of the nucleotide sequence, of the nucleic acid, see forexample, Zuker M., “Mfold web server for nucleic acid folding andhybridization prediction”, Nucleic Acids Res. 2003, 31, pp. 3406-3415.

Alternatively, the secondary structure is, predicted by preparing alibrary of mutagenized nucleotide sequences of a DNA aptamer (dopedlibrary), using it in SELEX, exhaustively analyzing sequences binding toa target, and determining a site at which base pairs are conserved (seee.g., Kimoto M., et al., “Generation of high-affinity DNA aptamer usingan expanded genetic alphabet” Nat. Biotechnol., 2013, 31, pp. 453-457).For the existing DNA aptamer, the secondary structure of the DNA aptamercan also be predicted by use of structure information thereon obtainedby X-ray crystallography or NMR.

The base length of the existing DNA aptamer is not particularly limited.The base length can be appropriately set within the range where the DNAaptamer can exert its capacity to bind to a target.

The existing DNA aptamer may comprise at least one base analogue and/ormodified base. The content of the base analogue and/or modified base inthe DNA aptamer of the present invention may be 20% or less, preferably15% or less, more preferably 10% or less, of the total number ofnucleotides constituting the nucleic acid aptamer.

The hairpin structure to be substituted in this step preferably has asize equivalent to that of the mini-hairpin structure after thesubstitution. In this context, the “equivalent size” means that thedifference in nucleotide length between the hairpin structure to besubstituted and the mini-hairpin structure after the substitution ispreferably 5 or less, for example, 4 or less, 3 or less, 2 or less, or 1or less, or the hairpin structure to be substituted and the mini-hairpinstructure after the substitution have the same nucleotide length. Sincethe mini-hairpin structure consists of 7 to 14 bases as described abovethe hairpin structure to be substituted preferably consists of, forexample, 7 to 19 bases.

The stem structure constituting the hairpin structure to be substitutedin this step may have one to three internal loop structures and/or oneto three bulge structures. In this context, the number of basesconstituting each internal loop structure in the stem structure is, forexample, 10 or less, preferably 6 or less, 5 or less, 4 or less, 3 orless, or 2, in the total of the two strands. The number of basesconstituting each bulge saliently in the stem structure is, for example,10 or less, preferably 6 or less, 5 or less, 4 or less, 3 or less, 2 orless, or 1.

As examples of this step, the substitutions of hairpin structurescomprising a stem structure comprising neither internal loop structurenor bulge structure, a stem structure comprising one internal loopstructure, or a stem structure comprising one bulge structure with aman-hairpin structure are shown in FIGS. 1(A), 1(B), and 1(C),respectively.

The substitution of the hairpin structure with the mini-hairpinstructure may be carried out by partially changing the nucleotidesequence constituting the hairpin structure or by completely changingthe nucleotide sequence constituting the hairpin structure.

When two or more hairpin structures are present in the existing DNAaptamer, these two or more hairpin structures may each be substitutedwith a mini-hairpin structure. For example, the whole hairpin structurescan be substituted therewith.

The hairpin structure to be substituted in this step is preferably ahairpin structure that is not involved in the binding of the DNA aptamerto the target molecule or makes a small contribution to the bindingthereof to the target molecule. Those skilled in the art can readilyidentify the hairpin structure that is not involved in the binding ofthe DNA aptamer to the target molecule or makes a small contribution tothe binding thereof to the target molecule, for example, by conductingX-ray crystallography or NMR analysis on a complex of the DNA aptamerand the target molecule. In the case of a DNA aptamer obtained by ascreening method such as the modified version of SELEX, the hairpinstructure that is not involved in the binding to the target molecule ormakes a small contribution to the binding to the target molecule canalso be predicted from a sequence conserved among selected DNA aptamers.For example, the hairpin structure that is not involved in the bindingto the target molecule or makes a small contribution to the binding tothe target molecule can be predicted by preparing a library ofmutagenized nucleotide sequences of a DNA aptamer (doped library), usingit in SELEX, exhaustively analyzing sequences binding to a target, anddetermining a site at which base pairs are conserved.

2-2. Step 2

In the present specification, the “step 2” is the step of increasing GCpairs in at least one stem structure, when the existing DNA aptamer hastwo or more stem structures. This step can be carried out bysubstituting one or more AT pairs in the stem structure with GC pairsand/or adding GC pairs into the stem structure. The site of the stemstructure of interest of the step 2 is not particularly limited as longas this site is not contained in the stem structure constituting themini-hairpin structure. The number of GC pairs to be increased is, forexample, 10 pairs or less, preferably 5 pairs or less, 4 pairs or less,3 pairs or less, 2 pairs or less, or 1 pair. For example, in the case ofthe substitution, all of AT pairs in the stem structure may besubstituted with GC pairs, or, for example, 10 pairs or less, preferably5 pairs or less, 4 pairs or less, 3 pairs or less, 2 pairs or less, or 1pair, of the AT pairs in the stem structure may be substituted with GCpairs. Likewise, in the case of adding GC pairs into the stem structure,for example, 10 pairs or less, preferably 5 pairs or less, 4 pairs orless, 3 pairs or less, 2 pairs or less, or 1 pair, of the GC pairs maybe added into the stem structure. The site to which GC pairs are addedmay be the end of the stem structure or may be an internal region of thestem structure.

When the DNA aptamer comprises a bulge structure or an internal loopstructure in the stem structure, in addition to or instead ofsubstituting one or more AT pairs in the stem structure with GC pairsand/or adding GC pairs into the stem structure, GC pairs in the stemstructure may be increased by subjecting bases constituting the bulgestructure or the internal loop structure to such substitution and/oraddition. Hereinafter, 4 separate cases will be described: (i) the casewhere the bulge structure comprises a base G or C, (ii) the case wherethe bulge structure comprises, a base A or T, (iii) the case where thenumber of nucleotides constituting the internal loop structure is equalbetween one strand and another strand, and (iv) the case where thenumber of nucleotides constituting the internal loop structure differsbetween one strand and another strand.

(i) Case where Bulge Structure Comprises Base G or C

In this case, GC pairs in the stem structure may be increased by addingG or C to the other strand so that that added base(s) are base-pairedwith base(s) constituting the bulge structure. The same number of basesas that of bases constituting the bulge structure may be added to theother strand. In this case, after the base addition, bases of the twostrands constituting the stem structure are completely paired so thatthe bulge structure no longer exists. A smaller number of bases thanthat of bases constituting the bulge structure may be added to the otherstrand. In this case, after the base addition, bases of the two strandsconstituting the stem structure are partially paired so that the bulgestructure remains. The number of G or C to be added is not particularlylimited as long as a smaller number of G or C than that of basesconstituting the bulge structure is added. The number of G or C to beadded is, for example, 10 less, preferably 5 or less, 4 or less, 3 orless, 2 or less, or 1 or less.

One example of adding the same number of bases as that of basesconstituting the bulge structure to the other strand and one example ofadding a smaller number of bases than that of bases constituting thebulge structure to the other strand are shown in FIGS. 2(A) and 2(B),respectively.

(ii) Case where Bulge Structure Comprises Base A or T

In this case, GC pairs in the stem structure may be increased bysubstituting A or T constituting the bulge structure with G or C, andthen adding G or C to the other strand so that that added base(s) arebase-paired with the substituted base(s). After the substitution of A orT constituting the bulge structure with G or C, G or C is added inaccordance with the case (i). All of bases A or T constituting the bulgestructure may be subjected to such substitution and addition, or aportion of the bases A or T may be subjected to the substitute andaddition.

One example of this step is shown in FIG. 3.

(iii) Case where the Number of Nucleotides Constituting Internal LoopStructure is Equal Between One Strand and Another Strand

In this case, GC pairs in the stem structure may be increased bysubstituting the same numbers of bases, for example, 10 bases or less,preferably 5 bases or less, 4 bases or less, 3 bases or less, 2 bases orless, or 1 base each, of the two strands constituting the internal looppresent in the stem structure with GC pairs. All of bases constitutingthe internal loop may be substituted with GC pairs, or a portion of thebases may be substituted with GC pairs. When a portion of the bases aresubstituted with GC pairs, this substitution with GC pairs preferablyoccurs at the end of the internal loop structure, i.e. the portion atwhich the internal loop is in contact with the stem structure. When thissubstitution with GC pairs occurs within the internal loop structure,i.e., a portion in the internal loop with which the stem structure isnot in contact, an additional bulge structure and/or internal loopstructure may be formed in the stem structure. In this case, the newlyformed bulge structure and/or internal loop structure may be subjectedto substitution and/or addition in accordance with the cases (i) to (iv)to further increase GC pairs in the stem structure.

One example of substituting all of bases constituting the internal loopwith GC pairs, one example of substituting a portion of the bases withGC pairs, wherein this substitution occurs at the end of the internalloop structure, and one example of substituting a portion of the baseswith GC pairs, wherein this substitution occurs within the internal loopstructure are shown in FIGS. 4(A), 4(B), and 4(C), respectively.

(iv) Case where the Number of Nucleotides Constituting Internal LoopStructure Differs Between One Strand and Another Strand

As in the case (iii), GC pairs in the stem structure may be increased bysubstituting the same numbers of unpaired bases, for example, 10unpaired bases or less, preferably 5 impaired bases or less, 4 unpairedbases or less, 3 unpaired bases or less, 2 unpaired bases or less, or 1unpaired base each, of the two strands of the stem structure with GCpairs. The substitution site is the same as in the case (iii), so thatthe description is omitted. In the case (iv), however, a bulge structureis finally formed in the one strand by increasing the same numbers ofsubstitutions of unpaired bases in the two strands with GC pairs. Inthis case, the formed structure may be subjected to substitution and/oraddition in accordance with the case (i) or (ii) to further increase GCpairs in the stem structure.

One example of this step is shown in FIG. 5.

2-3. Step 3

In the present specification, the “step 3” is the step of increasing GCpairs at the end of the DNA aptamer, when at least one end of the DNAaptamer forms a stem structure. The number of GC pairs to be added isnot limited as long as the binding activity of the resulting DNA aptameragainst the target molecule is not reduced. The number of GC pairs to beadded is, for example, 5 pairs or less, preferably 3 pairs, or less, 2pairs or less, 1 pair.

This step can be carried out by adding GC pair(s), i.e., G and C or Cand G, to the 5′ end and the 3′ end, respectively. When one strand ofthe stem structure constituting the end of the DNA aptamer has aprotruding end comprising base(s) G or C, this step can be carried outby adding G or C to the other strand so as to form base pairing with thebase(s) constituting this protruding end. When one strand of the stemstructure constituting the end of the DNA aptamer has a protruding endcomprising base(s) A or T, this step can be carried out by substitutingthis base(s) with G or C and then adding G or C to the other strand soas to form base pairing with the base(s) constituting this protrudingend.

2-4. Step 4

In the present specification, the “step 4” is the step of adding themini-hairpin structure to one end of the DNA aptamer. Whether themini-hairpin structure is added to the 5′ end or the 3′ end is notparticularly limited. The mini-hairpin structure is preferably added tothe 3′ end.

2-5. Effect

The method for designing a nucleotide sequence according to the presentinvention can enhance the capacity of an existing DNA aptamer to bind toa target molecule and/or stabilize the aptamer.

In the present specification, the “capacity to bind to a targetmolecule” means the ability to bind to a target molecule. An enhancedcapacity to bind to a target molecule can improve the ability of the DNAaptamer to specifically inhibit or suppress the functions (e.g.,biological activity) of the target molecule.

In the present specification, the “stabilization” means increase instability against heat and/or increase in stability against nucleolyticenzymes. An enhanced stability against heat and stability againstnucleolytic enzymes can enhance in vivo stability.

3. Method for Producing a DNA Aptamer Being Stabilized and/or HavingEnhanced Capacity to Bind to Target Molecule

According to the second embodiment, the present invention relates to amethod for producing a DNA aptamer being stabilized and/or havingenhanced capacity to bind to a target molecule, comprising the steps of:designing a nucleotide sequence of a DNA aptamer in accordance with themethod for designing a nucleotide sequence according to the firstembodiment; and producing the DNA aptamer on the basis of the designednucleotide sequence. The step of designing is as described in the firstembodiment, so that the description is omitted here.

In the present specification, the “DNA aptamer being stabilized and/orhaving enhanced capacity to bind to a target molecule” means a DNAaptamer that has stronger capacity to bind to a target molecule and/oris more stabilized than the existing DNA aptamer used in the method fordesigning a nucleotide sequence according to the first embodiment.

The step of producing a DNA aptamer is not particularly limited. Amethod known in the art may be used. For example, the DNA aptamer of thepresent invention may be chemically synthesized in accordance with aknown solid-phase synthesis method on the basis of the designed sequenceof the DNA aptamer as described above. For the chemical nucleic acidsynthesis method, see, for example, Current Protocols in Nucleic AcidChemistry, Volume 1, Section 3. As for such chemical synthesis, manylife science manufacturers (e.g., Takara Bio Inc., Life TechnologiesCorporation, and Sigma-Aldrich Corporation) provide contractmanufacturing services, and these services may be used. Severalfragments may be synthesized on the basis of the designed sequence ofthe DNA aptamer and then linked by intramolecular annealing or ligationor the like using ligase to prepare the DNA aptamer.

The DNA aptamer of the present invention thus chemically synthesized ispreferably purified by a method known in the art before use. Examples ofthe purification method include gel purification, affinity columnpurification, HPLC methods, and the like.

4. DNA Aptamer

According to the third embodiment, the present invention relates to aDNA aptamer comprising at least one stem structure and loop structure,wherein a hairpin structure located in a region other than the end ofthe DNA aptamer is the mini-hairpin structure. The DNA aptamer of thepresent invention may be obtained by the production method described inthe second embodiment.

In the present specification, the “region other than the end” is notparticularly limited as long as the region is any region of the DNAaptamer except for the 5′ end and the 3′ end. The region other than theend of the DNA aptamer is, for example, a site preferably 2 bases ormore, for example, 3 bases, 4 bases, or 5 bases or more, distant fromthe end portion of the DNA.

The DNA aptamer of the present invention may further comprise themini-hairpin structure at one end, in addition to the mini-hairpinstructure in the region other than the end. Whether the DNA aptamercomprises the mini-hairpin structure at 3′ end or 5′ end is not limited.Particularly preferably, the DNA aptamer comprises the mini-hairpinstructure at the 3′ end. The DNA aptamer of the present invention mayfurther have at least one stem structure and/or at least one loopstructure, in addition to the mini-hairpin structure. In this case, thestem structure of the DNA aptamer may internally have at least one basemismatch site or at least one bulge structure.

The base length of the DNA aptamer of die present invention is notparticularly limited. The base length can be appropriately set withinthe range where the DNA aptamer can exert its functions.

The DNA aptamer of the present invention may comprise at least one baseanalogue and/or modified base. The content of the base analogue and/ormodified base in the DNA aptamer of the present invention may be 20% orless, preferably 15% or less, more preferably 10% or less, of the totalnumber of nucleotides constituting the nucleic acid aptamer.

The DNA aptamer of the present invention preferably has a high GCcontent in at least one, for example, one, two, three, or all stemstructures. The GC content means the ratio of GC pairs to all base pairsconstituting stem structures contained in the DNA aptamer. The GCcontent in at least one, for example, one, two, three, or an stemstructures of the DNA aptamer of the present invention is, for example,50% or more, 75% or more, or 90% or more. All base pairs in at leastone, for example, one, two, three, or all stem structures may be GCpairs. When the end of the DNA aptamer of the present invention has astem structure, the terminal base pair is preferably a GC pair.

5. DNA Aptamer for IFN-γ

The fourth embodiment of the present invention relates to a DNA aptamerfor interferon-γ (abbreviated to “IFN-γ” in the present specification).The DNA aptamer for IFN-γ of the present invention has the constitutionof the DNA aptamer described in the third embodiment.

The DNA aptamer for IFN-γ of the present invention is a DNA aptamercomprising a single-stranded DNA molecule that strongly and specificallybinds to IFN-γ as a target substance and inhibits the cytotoxic Tcell-inducing activity of IFN-γ (hereinafter, this DNA aptamer isreferred to as a “DNA aptamer for IFN-γ”). The organism species fromwhich the target molecule IFN-γ of the DNA aptamer for IFN-γ of thepresent invention is derived is not limited. Examples thereof includeIFN-γ from mammals, for example, primates such as humans and chimpanzee,laboratory animals such as rats and mice, livestock animals such aspigs, cattle, horses, sheep, and goats, and pet animals such as dogs andcats, preferably humans.

The DNA aptamer for IFN-γ of the present invention consists of thenucleotide sequence as shown in any of SEQ ID NOs: 6 and 8 to 11.Particularly, the DNA aptamer for IFN-γ of the present inventionconsists of the nucleotide sequence as shown in SEQ ID NO: 8 or 9.

6. DNA Aptamer for VEGF

The fifth embodiment of the present invention relates to a DNA aptamerfor vascular endothelial growth factor (abbreviated to “VEGF” in thepresent specification).

VEGF is a growth factor that functions as an angiogenic factor and isknown as a causative factor of age-related macular degeneration (AMD).

The age-related macular degeneration is a progressive retinal diseasethat brings about severe symptoms such as decreased visual performanceor acquired blindness in adult humans. This disease has been found to beaggravated and to be severe with progress in angiogenesis in the retina(Martin A. et al., 2003, Medicinal Research Reviews, Vol. 23, No. 2:117-145; and Ferris III, F. L. et al., 1984, Archives of Ophthalmology,Vol. 102, Issue 11: 1640-1642).

The DNA aptamer for VEGF of the present invention is a DNA aptamercomprising a single-stranded DNA molecule that strongly and specificallybinds to VEGF as a target substance and inhibits the angiogenic functionof VEGF (hereinafter, this DNA aptamer is referred to as a “DNA aptamerfor VEGF”). The organism species from which the target molecule VEGF ofthe DNA aptamer for VEGF of the present invention is derived is notlimited. Examples thereof include VEGF from mammals, for example,primates such as humans and chimpanzee, laboratory animals such as ratsand mice, livestock animals such as pigs, cattle, horses, sheep, andgoats, and pet animals such as dogs and cats, preferably humans.

The DNA aptamer for VEGF of the present invention consists of thenucleotide sequence as shown in any of SEQ ID NOs: 19 to 22.Particularly, the DNA aptamer for VEGF of the present invention consistsof the nucleotide sequence as shown in SEQ ID NO: 20 or 22.

7. DNA Aptamer for vWF

The sixth embodiment of the present, invention relates to a DNA aptamerfor von Willebrand factor (abbreviated to “vWF” in the presentspecification), particularly, a DNA aptamer for vWF A1 domain.

The DNA aptamer for vWF of the present invention is a DNA aptamercomprising a single-stranded DNA molecule that strongly and specificallybinds to vWF, particularly, vWF A1 domain, as a target substance(hereinafter, this DNA aptamer is referred to as a “DNA aptamer forvWF”). The organism species from which the target molecule vWF of theDNA aptamer for vWF of the present invention is derived is not limited.Examples thereof include vWF from mammals, for example, primates such ashumans and chimpanzee, laboratory animals such as rats and, mice,livestock, animals such as pigs, cattle, horses, sheep, and goats, andpet animals such as dogs and cats, preferably humans.

The DNA aptamer for vWF, particularly, vWF A1 domain, of the presentinvention consists of the nucleotide sequence as shown in any of SEQ IDNOs: 14 to 16. Particularly, the DNA aptamer for vWF, particularly, vWFA1 domain, of the present invention consists of the nucleotide sequenceas shown in SEQ ID NO: 16.

8. Pharmaceutical Composition

The seventh embodiment of the present invention relates to apharmaceutical composition.

8-1. Constitution

The pharmaceutical composition of the present invention comprises atleast one DNA aptamer described in any of the third to sixthembodiments. The pharmaceutical composition of the present invention mayalso contain a pharmaceutically acceptable carrier. The“pharmaceutically acceptable carrier” refers to a substance that isusually used in the pharmaceutical formulating art and added withoutinhibiting or suppressing the effects of the pharmaceutical compositionin order to facilitate the formulation of the pharmaceutical compositionor its application to organisms and maintain the effect of the inhibitorof target substance function. Examples of the carrier includeexcipients, binders, disintegrants, fillers, emulsifiers, flow controladditives, lubricants, and surfactants.

Examples of the “excipients” include sugars such as monosaccharides,disaccharides, cyclodextrin, and polysaccharides (specificallyincluding, but not limited to, glucose, sucrose, lactose, raffinose,mannitol, sorbitol, inositol, dextrin, maltodextrin, starch, andcellulose), metal salts (e.g., sodium phosphate or calcium phosphate,calcium sulfate, and magnesium sulfate), citric acid, tartaric acid,glycine, low-, middle-, or high-molecular-weight polyethylene glycol(PEG), Plutonic, and combinations thereof.

Examples of the “binders” include starch glues composed of corn, wheat,rice, or potato starch, gelatin, tragacanth, methylcellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone.

Examples of the “disintegrants” include the starches described above,carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, alginicacid or sodium alginate, and salts thereof.

Examples of the “fillers” include the sugars described above and/orcalcium phosphate (e.g., tricalcium phosphate or calcium hydrogenphosphate).

Examples of the “emulsifiers” include sorbitan fatty acid ester,glycerin fatty acid ester, sucrose fatty acid ester, and propyleneglycol fatty acid ester.

Examples of the “flow control additives” and the “lubricants” includesilicate, talc, stearate, and polyethylene glycol.

Such carriers may be used appropriately according to the need. Thepharmaceutical composition of the present invention may also contain, inaddition to the additives described above, optional additives such ascorrigents, solubilizing agents (solubilizers), suspending agents,diluents, surfactants, stabilizers, absorption promoters (e.g.,quaternary ammonium salts and sodium lauryl sulfate), expanders, wettingagents, humectants (e.g. glycerin and starch), adsorbents (e.g., starch,lactose, kaolin, bentonite, and colloidal silicic acid), disintegrationinhibitors (e.g., saccharose, stearin, cacao butter, and hydrogenatedoil), coating agents, coloring agents, preservatives, antioxidants,fragrances, flavors, sweeteners, and buffers.

The “surfactants” correspond to, for example, alkali metal salts,alkaline earth metal salts, and ammonium salts of lignosulfonic, acid,naphthalenesulfonic acid, phenolsulfonic acid, ordibutylnaphthalenesulfonic acid, alkylaryl sulfonate, alkyl sulfate,alkyl sulfonate, fatty alcohol sulfate, fatty acid and sulfated fattyalcohol glycol ether, condensates of sulfonated naphthalene ornaphthalene derivatives and formaldehyde, condensates of naphthalene ornaphthalenesulfonic acid, phenol, and formaldehyde, polyoxyethyleneoctylphenyl ether, ethoxylated isooctylphenol, octylphenol, nonylphenol,alkylphenyl polyglycol ether, tributylphenyl polyglycol ether,tristearylphenyl polyglycol ether, alkylaryl polyether alcohol, alcoholand fatty alcohol/ethylene oxide condensates, ethoxylated castor oil,polyoxyethylene alkyl ether, ethoxylated polyoxypropylene, laurylalcohol polyglycol ether acetal, sorbitol ester, lignosulfite wasteliquors, and methylcellulose.

The pharmaceutical composition of this embodiment may contain one ormore of these carriers per pharmaceutical composition.

The pharmaceutical composition of the present invention may furthercontain an additional drug without canceling the pharmacological effectof the nucleic acid of the present invention. The pharmaceuticalcomposition of the present invention may contain, for example, aspecific amount of an antibiotic.

The dosage form of the pharmaceutical composition of the presentinvention is not particularly limited as long as the form does notdeactivate the active ingredient and can exert the pharmacologicaleffect in vivo after administration. The dosage form usually variesdepending on an administration method and/or prescription conditions.

Examples of dosage forms suitable for oral administration can includesolid preparations (including tablets, pills, sublingual preparations,capsules, drops, and troches), granules, dusts, powders, and liquidpreparations. The solid preparations can be prepared, if necessary, incoated dosage forms known in the art, for example, as sugar-coatedtablets, gelatin-coated tablets, enteric coated tablets, film-coatedtablets, bilayer tablets, or multilayer tablet.

Parenteral administration is subdivided into systemic administration andlocal administration. The local administration is further subdividedinto interstitial administration, transepidermal administration,transmucosal administration, and transrectal administration. Thepharmaceutical composition may also be prepared in a dosage formsuitable for each administration method. Examples of dosage formssuitable for systemic or interstitial administration include injectionswhich are liquid preparations. Examples of dosage forms suitable fortransepidermal administration or transmucosal administration can includeliquid preparations (including liniments, eye drops, nasal drops, andinhalants), suspensions (including emulsions and creams), dusts(including nasal drops and inhalants), pastes, gels, ointments, andplasters. Examples of dosage forms suitable for transrectaladministration can include suppositories.

In the case of drug administration to plants, examples of the dosageform of the pharmaceutical composition include liquids, solids(including semi-solids), and combinations thereof. In this case, thepharmaceutical composition may be prepared as solutions, oildispersions, emulsions, suspensions, dusts, powders, pastes, gels,pellets, tablets, and granules.

The specific shapes or sizes of these dosage forms are not particularlylimited and may be any shape or size that falls within ranges acceptedfor each dosage form known in the art.

8-2. Production Method

The pharmaceutical composition of the present invention may be producedby the application of a formation method known in the art, as a rule.See a method described in, for example, Remington's PharmaceuticalSciences (Merck Publishing Co., Easton, Pa.).

For example, the injection may be produced by a method routinely used inthe art which involves dissolving the DNA aptamer of any of the third tosixth embodiments in a pharmaceutically acceptable solvent and, ifnecessary, adding a pharmaceutically acceptable carrier to the resultingsolution.

Examples of the “pharmaceutically acceptable solvent” include water,ethanol, propylene glycol, ethoxylated isostearyl alcohol,polyoxygenated isostearyl alcohol, and polyoxyethylene sorbitan fattyacid esters. Desirably, such a solvent is sterilized and, if necessary,preferably adjusted to be isotonic to blood.

8-3. Administration Method

The pharmaceutical composition of this embodiment may be administered toan organism in a pharmaceutically effective amount for the treatment orprevention of the disease of interest or the like. The recipientorganism is a vertebrate, preferably a mammal, more preferably a human.

The pharmaceutical composition of the present invention may beadministered systemically or locally. An appropriate route can beselected according to, for example, the type, site of onset, or degreeof progression of the disease. For a disease whose onset is localized toa site, local administration is preferred in which the pharmaceuticalcomposition of the present invention is directly administered to thesite of onset and its neighborhood through injection or the like. Thisis because the DNA aptamer of the present invention can be delivered insufficient amounts to the site (tissue or organ) to be treated withlittle influence on the other tissues. For a disease whose site to betreated cannot be identified or a disease whose onset is systemic,systemic administration through intravenous injection or the like ispreferred, though the administration route is not limited thereto. Thisis because the DNA aptamer of the present invention can be distributedthroughout the body via blood flow and thereby delivered even to alesion that cannot be found by diagnosis.

The pharmaceutical composition of the present invention may beadministered by any appropriate method without deactivating the activeingredient. For example, any of parenteral (e.g., injection, aerosol,application, eye drop, and nasal drop) and oral administrations may beperformed. Injection is preferred.

In the case of administration through injection, an injection site isnot particularly limited. The injection site may be any site at whichthe DNA aptamer serving as an active ingredient can bind to the targetsubstance to suppress its functions. Examples thereof includeintravenous, intraarterial, intrahepatic, intramuscular, intraarticular,intramedullary, intraspinal, intraventricular, transpulmonary,transdermal, hypodermic, intradermal, intraperitoneal, intranasal,enteral, and sublingual injections, intravascular injection such asintravenous injection or intraarterial injection is preferred. This isbecause, as mentioned above, the pharmaceutical composition of thepresent invention can be distributed throughout the body via blood flowand also because this injection is relatively low invasive.

9. Method for Detecting Target Substance

The eighth embodiment of the present invention relates to a method fordetecting a target substance using the DNA aptamer described in any ofthe third to sixth embodiments.

9-1. Constitution

The DNA aptamer described in any of the third to sixth embodiments iscapable of very strongly and specifically binding to its targetsubstance. The target substance present in a sample can therefore bedetected by use of this property of the DNA aptamer.

The detection method itself can be any detection method known in the artas long as the method is based on the binding between the DNA aptamerdescribed in any of the third to sixth embodiments and the targetsubstance. For example, a SPR method, a quartz crystal microbalancemethod, turbidimetry, colorimetry, or fluorometry may be used.

SPR (surface plasmon resonance) refers to a phenomenon in which as athin metal film is irradiated with laser beam, reflected light intensityremarkably attenuates at a particular angle of incidence (resonanceangle). The SPR method is an assay method based on this phenomenon andis capable of highly sensitively assaying a substance adsorbed on thesurface of the thin metal film serving as a sensor portion. In thepresent invention, for example, the target substance in the sample maybe detected by immobilizing the DNA aptamer of any of the third to sixthembodiments in advance onto the surface of a thin metal film, flowingthe sample on the thin metal film surface, and detecting the differencein the substance adsorbed on the metal surface between before and afterthe sample flowing resulted from the binding between the DNA aptamer andthe target substance. SPR methods such as a displacement method and anindirect competitive method are known, any of which may be used in thepresent invention.

The QCM (quartz crystal microbalance) method refers to a method using aphenomenon in which the resonance frequency of a quartz crystaldecreases according to the mass of the substance adsorbed onto thesurface of electrodes attached to the quartz crystal. A QCM sensor basedon this method can quantitatively capture a trace amount of the adsorbedsubstance according to the amount of change in the resonance frequencyof a quartz crystal. In the present invention, the target substance inthe sample can be quantitatively detected based on the amount of changein the resonance frequency of a quartz crystal resulted from the bindingbetween the DNA aptamer and the target substance, by immobilizing theDNA aptamer advance, as in the SPR method, onto the electrode surface,and contacting a sample with the electrode surface. This technique iswell known in the art. See, for example, Christopher J., et al. (2005),Self-Assembled Monolayers of a Form of Nanotechnology, Chemical Review,105: 1103-1169.

The turbidimetry refers to a method which involves irradiating asolution with light and optically measuring the attenuation of lightscattered by a substance floating in the solution or light transmittedthrough the solution using a colorimeter or the like to determine theamount of the substance in the solution. In the present invention, thetarget substance in the sample can be quantitatively detected bymeasuring absorbance before and after addition of the DNA aptamer of anyof the third to sixth embodiments into a sample.

Alternatively, the target substance may be detected by combined use withan antibody against the target substance. For example, a method based onsandwich ELISA may be used. This method involves first immobilizing theDNA aptamer of any of the third to sixth embodiments onto a solid-phasecarrier and next adding a sample thereto to allow the DNA aptamer tobind to the target substance present in the sample. Subsequently, thesample is washed off. Then, the anti-target substance antibody is addedthereto and allowed to bind to the target substance. After washing, thetarget substance in the sample may be detected by detecting theanti-target substance antibody using an appropriately labeled secondaryantibody. An insoluble carrier in the form of, for example, beads, amicroplate, a test tube, a stick, or a test piece made of a materialsuch as polystyrene, polycarbonate, polyvinyltoluene, polypropylene,polyethylene, polyvinyl chloride, nylon, polymethacrylate, latex,gelatin, agarose, cellulose, Sepharose, glass, a metal, a ceramic, or amagnetic material can be used as the solid-phase carrier.

EXAMPLES Example 1: Preparation of DNA Aptamer Containing Mini-HairpinSequence

In order to examine whether the introduction of a mini-hairpin structure(short DNA fragment consisting of a sequence such as GCGNNACGC (SEQ IDNO: 23) or GCGNAGC (SEQ ID NO: 24) (N=A, T, G, or C)) and thesubstitution of A-T base pairs in a stem sequence with G-C base pairsare effective for the stability of a DNA aptamer, various nucleic acidfragments were first designed and prepared for DNA aptamers for humanIFN-γ. The sequences of the aptamers prepared in this Example and theirsecondary structures are shown an FIG. 6.

Aptamer 49 (SEQ ID NO: 4) is a previously reported IFN-γ-binding DNAaptamer found by the inventors. Aptamer 49A (SEQ ID NO: 5) was derivedfrom Aptamer 49 by the substitution of the artificial base Ds with thenatural base A. Aptamer 58 (SEQ ID NO: 6) was derived from Aptamer 49 bythe introduction of a mini-hairpin structure (short DNA fragmentconsisting of the sequence CGCGAAGCG (SEQ ID NO: 1)) to the 3′ end.Aptamer 57mh (SEQ ID NO: 9) was derived from Aptamer 58 by thesubstitution of the internal hairpin of the sequence with a mini-hairpinstructure (short DNA fragment consisting of the sequence CGCGAAGCG (SEQID NO: 1)). Aptamer 58GC (SEQ ID NO: 7) and Aptamer 57GCmh (SEQ ID NO:8) were derived from Aptamer 58 and Aptamer 57mh by the substitution ofA-T base pairs at two sites in the stem structure with G-C base pairs.

For Aptamer 49, Aptamer 58, Aptamer 58GC, Aptamer 57mh, Aptamer 57GCmh,and Aptamer 49A, each nucleic acid having the nucleotide sequence shownin the diagram was prepared by chemically synthesis and then purified ona denaturing acrylamide gel. Each nucleic acid fragment was heated at95° C. for 3 minutes in a phosphate buffer solution (pH 7.4), then leftstanding at room temperature for 10 minutes for slow cooling, and leftstanding for 5 minutes on ice for reconstruction to prepare the DNAaptamer.

Example 2: Analysis on Binding Activity of Each Aptamer for IFN-γAgainst Human IFN-γ

In order to examine each prepared nucleic acid prepared in Example 1 forits binding to human IFN-γ, a competition test was conducted usingAptamer 49 containing the artificial base Ds. In 20 μL of a reactionsolution (1×PBS and 0.005% Nonidet P-40), Aptamer 49 labeled with[γ-³²P]ATP (final concentration: 200 nM), the aptamers prepared inExample 1 (final concentration 200 nM), and human IFN-γ (20 nM,PeproTech, Inc.) were mixed and incubated at 37° C. for 30 minutes.Then, 25% glycerol containing bromophenol blue was added thereto at afinal glycerol concentration of 5%. Labeled Aptamer 49 bound to humanIFN-γ was separated from labeled Aptamer 49 in a free form by 10%non-denaturing polyacrylamide gel electrophoresis. The gel was dried andvisualized using Bio Image Analyzer FLA-7000 (Fujifilm), and theradioactivity was measured. The gel shift rate, the inhibition rate, andthe relative binding rate were calculated as follows. The gel shift ratewas calculated as the percentage of the value obtained by dividing theradioactivity of the complex by the total radioactivity of the free formand complex. The inhibition rate was calculated as the value obtained bysubtracting, from 100, the percentage of the value obtained by dividingthe gel shift rate in the presence of a competing aptamer by the gelshift rate in the absence of a competing aptamer. The relative bindingrate was calculated by dividing the inhibition rate of each aptamer bythe inhibition rate of Aptamer 49.

Three aptamers (Aptamer 49, Aptamer 58, and Aptamer 57GCmh) were furtherpicked up from among the sequences prepared in Example 1 and theircapacity to bind to human IFN-γ were analyzed by surface plasmonresonance (SPR) measurement using BIAcore T200 (GE Healthcare JapanCorp.).

First, a nucleic acid fragment having each sequence biotinylated at theend of the structure was chemically synthesized. In the same way as inExample 1, each nucleic acid fragment was then purified on a denaturingacrylamide gel, heated at 95° C. in a phosphate buffer solution (pH7.4), and then slowly cooled to 25° C. for reconstruction to prepare theDNA aptamer. The SPR sensor chip used was a streptavidin-coated SA Chip(GE Healthcare Japan Corp.). Each DNA fragment was irreversiblyimmobilized onto the chip by a method given below and then analyzed forits binding to human IFN-γ. SPR was measured at a temperature set to 25°C. using a running buffer (1×PBS, +50 mM NaCl (final NaCl concentration:205 mM), and 0.05% Nonidet P-40). For the immobilization of each DNAfragment onto the sensor chip, a DNA solution diluted with a PBSsolution to 25 nM was subjected to folding treatment (denaturation byheating at 95° C. for 3 minutes followed by slow cooling to 25° C.), anddiluted to 0.5 nM in tuning buffer, and Nonidet P-40 was then addedthereto at a final concentration of 0.05%, and this DNA solution (40 μL)was injected to the SA chip at a flow rate of 5 μL/min (corresponding to8 min). After the immobilization, DNA fragments nonspecifically adsorbedon the SA chip were washed off by the injection (5 μL×5) of a 50 mM NaOHsolution at a flow rate of 20 μL/min. The interaction between theimmobilized DNA fragment and human IFN-γ was detected under monitoringby the injection of 1.25 nM, 2.5 nM, 5 nM, 10 nM, 20 nM, 30 nM, and 50nM human IFN-γ solutions (diluted with a running buffer) at the KineticInjection mode. The measurement conditions involved a flow rate of 100μL/min and a protein injection time of 150 seconds. The regeneration ofthe chip (dissociation of bound proteins and DNA refolding) wasperformed by the injection of 5 μL (corresponding to 15 sec) of a 50 mMNaOH solution followed by the injection of a running buffer for 10minutes. In order to cancel bulk effect on the sensor chip or responsevalues attributed to nonspecific adsorption, the response value of aDNA-unimmobilized cell used as a reference cell was subtracted from thesensorgram of each DNA fragment.

<Results>

The binding inhibition ratio of each aptamer variant to Aptamer 49 isshown in FIG. 7. When the binding inhibition ratio of the controlAptamer 49 was defined as 1.00, the relative binding rate of eachaptamer variant containing the mini-hairpin structure was 0.05 (Aptamer49A), 1.29 (Aptamer 58), 0.24 (Aptamer 58GC), 1.53 (Aptamer 57GCmh), and1.40 (Aptamer 57mh). Aptamer 58, Aptamer 57mh, and Aptamer 57GCmhcontaining the mini-hairpin were shown to have significantly highbinding strength, as compared with the control Aptamer 49. Among them,Aptamer 57GCmh in which a mini-hairpin was added to the 3′ end, theinternal hairpin portion was substituted with a mini-hairpin, andfurther, A-T base pairs in stem portions were substituted with G-C basepans had particularly high binding strength.

The results of the SPR measurement are shown in FIG. 8. The KD value ofeach oligo was 46 pM (Aptamer 49), 38 pM (Aptamer 58), and 33 pM(Aptamer 57GCmh). These results demonstrated that binding strength canbe improved by adding mini-hairpin DNA to the 3′ end, furthersubstituting internal hairpin with mini-hairpin DNA, and furthersubstituting A-T base pairs in stem portions with G-C base pairs.

Example 3: Analysis on Stability of Each Aptamer for IFN-γ AgainstNucleolytic Enzyme, Etc.

<Method>

Nucleolytic enzymes contained in serum are one of the main causes of DNAdegradation, which hinders the biological application of nucleic acids.In this respect, each DNA aptamer containing the mini-hairpin structurewas examined for its stability against nucleolytic enzymes contained inhuman serum.

Each DNA aptamer (Aptamer 49 Aptamer 58, Aptamer 57mh, and Aptamer57GCmh; final concentration: 2 μM) was mixed with human serum at aconcentration of 96%, and the mixture was incubated at 37° C. After 0hours, 1 hour, 6 hours, 24 hours, 48 hours, and 72 hours, 10 μL wassampled from the mixed solution, and the degradation reaction wasterminated by mixing with 110 μL of 1×TBE and a 10 M Urea solution. Eachsample thus reacted was fractionated by 15% denaturing polyacrylamidegel electrophoresis. Then, the gel was stained with SYBR GOLD to detecta single-stranded nucleic acid. The band pattern of a product degradedby the nucleolytic enzymes in the human serum was analyzed using BioImager LAS-4000 (Fijifilm Corp). The amount of residual DNA waspredicted from the density of a band corresponding to an undegradedaptamer.

<Results>

The results are shown in FIG. 9. The band corresponding to thefull-length product of the control DNA aptamer (Aptamer 49) was degradedto 55% after 6 hours in the presence of human serum. The bandcorresponding to the full-length product was degraded to 14% after 24hours and further degraded to 9% after 72 hours. On the other hand, theDNA aptamers containing the mini-hairpin DNA (Aptamer 58, Aptamer 57mh,and Aptamer 57GCmh) were confirmed, from their band patterns, to stillretain 30% or more of the whole amount in the state of a full-lengthproduct even after 72 hours in the presence of human serum. Among them,80% or more product of Aptamer 57GCmh in which a mini-hairpin was addedto the 3′ end, the internal hairpin portion was substituted withmini-hairpin DNA, and further, A-T base pairs in stem portions weresubstituted with G-C base pairs maintained the full length even after 72hours in the presence of human serum, demonstrating that stabilityagainst nucleolytic enzymes, etc., contained in human serum isremarkably enhanced by, for example, the addition of mini-hairpin DNA.

Example 4: Analysis on Thermal Stability of Each Aptamer for IFN-γ

<Method>

The thermal stability of each DNA aptamer (Aptamer 49, Aptamer 49A,Aptamer 58, Aptamer 58GC, Aptamer 57mh, and Aptamer 57GCmh; finalconcentration: 2 μM) was studied by the measurement of a Tm value.Change in the absorbance of the DNA aptamer caused by the elevation oftemperature (0.5° C./min) was measured using an ultraviolet and visiblespectrophotometer UV-2450 (Shimadzu Corp.). The melting temperature (Tmvalue) was calculated from the first derivation thereof.

<Results>

The results are shown in FIG. 10. The control DNA aptamer (Aptamer 49)had a Tm value of 37.8° C., whereas the DNA aptamer containing noartificial base (Aptamer 49A) had a Tm value of 33.4° C. The Ds aptamervariants containing the mini-hairpin structure had a Tm value of 43.9°C. (Aptamer 58), 72.0° C. (Aptamer 58GC), 51° C. (Aptamer 57mh), and64.2° C. (Aptamer 57GCmh). These results demonstrated that the additionof mini-hairpin DNA to the DNA aptamer remarkably increases the Tm valueand improves the thermal stability. These results also demonstrated thatthe substitution of A-T base pairs in stem sequences with G-C base pairsfurther increases the Tm value.

Example 5: Analysis on Inhibitory Effect of Each Ds Aptamer on CulturedCell in Response to IFN-γ Stimulation

In this Example, the degree of phosphorylation of STAT-1 in response toIFN-γ stimulation was analyzed in the presence of each prepared DNAaptamer to examine the inhibitory effect of each aptamer on theinteraction between IFN-γ and IFN-γ receptor.

<Method>

1) Cell Culture

MDA-MB-231 cells were cultured using a DMEM medium (Dulbecco's MinimalEssential Medium, Corning cellgro) containing 10% fetal bovine serum(FBS, Atlanta Biologicals, Inc.) and supplemented with a 100× solutionof antibiotics (penicillin and streptomycin) and L-glutamine (Gibco/LifeTechnologies Corp.).

2) Stimulation Treatment with IFN-γ

For pretreatment of stimulation with IFN-γ, a DMEM medium containing 10%FBS and 10⁶ cells/mL of MDA-MB-231 cells was placed in a roundpolystyrene tube (Falcon/Becton, Dickinson and Company) and incubated at37° C. for 15 minutes. Then, the cells were harvested by centrifugationat 1200 g for 5 minutes, then resuspended in a DMEM medium containing 2ng/mL of a recombinant protein human IFN-γ (PeproTech, Inc.), andincubated at 37° C. for 10 minutes for stimulation with IFN-γ. Then, thecells were harvested by centrifugation, and then the stimulation wasterminated by washing once with PBS to prepare cells after FACSanalysis.

3) Stimulation Treatment with IFN-γ in Presence of Aptamer

In the same way as in the preceding paragraph 2), for pretreatment ofstimulation, a DMEM medium containing 10% FBS and 10⁶ cells/mL ofMDA-MB-231 cells was placed in a round polystyrene tube (Falcon/Becton,Dickinson and Company) and incubated at 37° C. for 15 minutes. Then, thecells were harvested by centrifugation at 1200 g for 5 minutes, thenresuspended in a medium containing serum and each aptamer, and incubatedovernight at room temperature or 37° C. On the following morning, IFNγwas added thereto, and the cells were incubated at 37° C. for 10 minutesfor stimulation. Then, the cells were harvested by centrifugation, andthen the stimulation was terminated by washing once with PBS to preparecells for FACS analysis.

4) Preparation of Cell for FACS Analysis

The cells thus stimulated with IFN-γ and washed with PBS were suspendedin 1 mL of a 2% paraformaldehyde solution (Electron Microscopy Sciences)and incubated at room temperature for 10 minutes. Then, the cells wereharvested by centrifugation. The harvested cells were resuspended in 1ml of 90% cooled methanol and incubated for 30 minutes to 60 minutes orovernight at 4° C. under dark conditions. The cells harvested bycentrifugation were washed twice with 0.5 mL of a buffer solution forFACS analysis (containing 1×PBS, pH 7.4, 0.1% sodium azide, and 0.1%BSA), then incubated for 30 minutes to 60 minutes at 4° C. under darkconditions in 0.1 mL of a buffer solution for FACS analysis containing7.5 μL of an anti-phosphorylated STAT-1 antibody solution (BD PhosflowPE mouse anti-Stat-1 pY701), and washed twice with 0.5 mL of a buffersolution for FACS analysis. The cells thus washed were suspended in 0.5mL of a buffer solution for FACS analysis and then analyzed by flowcytometry.

<Results>

As a result of reacting the MDA-MB-231 cells with IFN-γ at aconcentration of 2 ng/mL at 37° C. for 10 minutes and conducting FACSanalysis using an anti-phosphorylated STAT-1 antibody, evident increasein fluorescence intensity induced by the anti-phosphorylated STAT-1antibody in response to IFN-γ stimulation was confirmed (FIG. 11).

Next, Aptamer 49 and Aptamer 58 in which mini-hairpin DNA was added tothe 3′ end were used and each added at a nucleic acid concentration of100 ng/mL to MDA-MB-231 cells at room temperature or at a reactiontemperature of 37° C. After overnight incubation, the cells were reactedwith IFN-γ for 10 minutes and analyzed by FACS using ananti-phosphorylated STAT-1 antibody. At room temperature, Aptamer 49 andAptamer 58 were both confirmed to inhibit the phosphorylation of STAT1(FIG. 12A). This demonstrated that the IFN-γ stimulation inhibitoryactivity of an aptamer is maintained even if mini-hairpin DNA is addedto the 3′ end. At 37° C., Aptamer 49 was degraded by nucleolyticenzymes, etc., contained in the medium and thereby lost its IFN-γstimulation inhibitory effect, whereas Aptamer 58 was shown to maintainits IFN-γ stimulation inhibitory effect (FIG. 12B). From this, theaddition of mini-hairpin DNA was confirmed to remarkably improve thestability against nucleolytic enzymes contained in serum in a medium.

Next, each DNA aptamer (Aptamer 49, Aptamer 49A, Aptamer 58, Aptamer57mh, and Aptamer 57GCmh) and an existing DNA aptamer for IFN-γ as acontrol (Aptamer 26) were used and examined for their IFN-γ stimulationinhibitory effects. Each aptamer was added at a concentration of 100 to200 ng/mL and reacted overnight at 37° C., followed by FACS analysisusing an anti-phosphorylated STAT-1 antibody. The control Aptamer 26,and Aptamer 49 and Aptamer 49A to which no mini-hairpin DNA was addedhardly exhibited an IFN-γ stimulation inhibitory effect, whereas Aptamer58, Aptamer 57mh, and Aptamer 57GCmh to which mini-hairpin DNA was addedwere confirmed to exhibit an IFN-γ stimulation inhibitory effect.Particularly, Aptamer 57mh and Aptamer 57GCmh in which mini-hairpin DNAwas added to the 3′ end and the internal hairpin portion was substitutedwith mini-hairpin DNA remarkably maintained their IFN-γ stimulationinhibitory effects and were thus found to have the evidently improvedstability by the addition of mini-hairpin DNA (FIG. 13).

Example 6: Analysis on Binding Activity of Aptamer for IFN-γ Consistingof Natural Bases Against Human IFN-γ

In this Example, a DNA aptamer consisting of natural bases withoutcontaining artificial bases was examined for whether the addition ofmini-hairpin DNA and the addition of GC pairs to the stem structurecould also improve its binding activity.

<Method>

A nucleic acid having each nucleotide sequence shown in FIG. 14 wasprepared by chemical synthesis.

As described above, Aptamer 49 (SEQ ID NO: 4) is a previously reportedIFN-γ-binding DNA aptamer found by the inventors. Aptamer 49A (SEQ IDNO: 5) was derived from Aptamer 49 by the substitution of the artificialbase Ds with the natural base A. Aptamer 57Amh (SEQ ID NO: 11) wasderived from Aptamer 49A by the introduction of a mini-hairpin structureto the 3′ end and the substitution of the internal hairpin of thesequence with a mini-hairpin structure (short DNA fragment consisting ofthe sequence CGCGAAGCG (SEQ ID NO: 1)). Aptamer 57AGCmh (SEQ ID NO: 10)was derived from Aptamer 57Amh by the substitution of A-T base pairs attwo sites in the stem structure with G-C base pairs.

In the same way as in Example 1, each nucleic acid consisting of thenucleotide sequence described above was chemically synthesized,purified, and reconstructed to prepare each DNA aptamers.

In order to examine each prepared nucleic acid for its binding to humanIFN-γ, a competition test was conducted using Aptamer 49, which was awild-type aptamer containing the artificial base Ds. In 20 μL of areaction solution (1×PBS and 0.005% Nonidet P-40), Aptamer 49 labeledwith [γ-³²P]ATP (final concentration: 20 nM), a 1000-fold amount of eachunlabeled nucleic acid (Aptamer 49A, Aptamer 57AGCmh, and Aptamer 57Amh)(final concentration: 20 μM), and human IFN-γ (20 nM, PeproTech, Inc.)were mixed and incubated at 37° C. for 30 minutes. Then, 25% glycerolcontaining bromophenol blue was added thereto at a final glycerolconcentration of 5%. Labeled Aptamer 49 bound to human IFN-γ wasseparated from labeled Aptamer 49 in a free form by 10% non-denaturingpolyacrylamide gel electrophoresis. The gel was dried and visualizedusing Bio Image Analyzer FLA-7000, and the radioactivity was measured.The gel shift rate, the inhibition rate, and the relative binding ratewere calculated in accordance with Example 2.

<Results>

The binding inhibition ratio of each aptamer variant to Aptamer 49 isshown in FIG. 15. When the binding inhibition ratio of the controlAptamer 49A was defined as 1.00, the binding inhibition ratio (relativebinding rate) of each aptamer variant containing the mini-hairpinstructure was 3.00 (Aptamer 57AGCmh) and 3.09 (Aptamer 57Amh). Aptamer57AGCmh and Aptamer 57Amh containing the mini-hairpin were shown to havesignificantly improved binding strength, as compared with the controlAptamer 49A.

Example 7: Analysis on Thermal Stability of Aptamer Consisting ofNatural Bases for IFN-γ

<Method>

The thermal stability of each DNA aptamer consisting of natural bases(Aptamer 49A, Aptamer 57Amh, and Aptamer 57AGCmh; final concentration: 2μM) was studied by the measurement of a Tm value. Change in theabsorbance of the DNA aptamer caused by the elevation of temperature(0.5° C./min) was measured using an ultraviolet and visiblespectrophotometer UV-2450 (Shimadzu Corp). The melting temperature (Tmvalue) was calculated from the first derivation thereof.

<Results>

The results are shown in FIG. 16. The control DNA aptamer (Aptamer 49A)had a Tm value of 33.4° C., whereas the aptamer variants consisting ofnatural bases containing the mini-hairpin structure had a Tm value of45.4° C. (Aptamer 57Amh) and 61.6° C. (Aptamer 57AGCmh). These resultsdemonstrated that the addition of mini-hairpin DNA to the DNA aptamerremarkably increases the Tm value and improves the thermal stability.These results demonstrated that the addition of mini-hairpin and theaddition of GC improve the thermal stability of a DNA aptamer regardlessof the presence or absence of the artificial base Ds.

Example 8: Analysis on Binding Activity of Aptamer for vWF Against vWFA1 Domain

A DNA aptamer for a protein other than IFN-γ was examined for whetherthe addition of mini-hairpin DNA could improve its binding activity.

<Method>

A nucleic acid having each nucleotide sequence shown in FIG. 17 wasprepared by chemical synthesis.

ARC1172(wt) (SEQ ID NO: 12) is a known vWF A1 domain-binding DNAaptamer. ARC1172(wt)-iT (SEQ ID NO. 13) was derived from ARC1172(wt) bythe addition of inverted dT to the 3′ end, which is a conventionaltechnique of stabilizing nucleic acids. ARC1172-F (SEQ ID NO: 14) wasderived therefrom by the substitution of the internal hairpin of thesequence with a mini-hairpin structure (short DNA fragment consisting ofthe sequence GCCGAAGGC (SEQ ID NO. 2)). ARC1172-F-iT (SEQ ID NO: 15) wasderived from ARC1172-F by time addition of inverted dT to the 3′ end,which is a conventional technique of stabilizing nucleic acids.ARC1172-G (SEQ ID NO: 16) was derived from ARC1172-F by the introductionof a mini-hairpin structure (short DNA fragment consisting of thesequence CGCGAAGCG (SEQ ID NO: 1)) to the 3′ end.

In the same way as in Example 1, each nucleic acid consisting of thenucleotide sequence described above was chemically synthesized,purified, and reconstructed to prepare each DNA aptamers.

In order to examine each prepared nucleic acid for its binding to vWF A1domain, competition with ARC1172 reported as an aptamer binding to vWFA1 domain was tested using each variant nucleic acid. In 20 μL of areaction solution (1×PBS and 0.1 mg/mL BSA), ARC1172 labeled with[γ-³²P]ATP (final concentration: 100 nM), each unlabeled nucleic acid(ARC1172(wt), ARC1172(wt)-iT, ARC1172-F, ARC1172-F-iT, and ARC1172-G)(final concentration: 100 nM), and vWF A1 domain (100 nM, U-ProteinExpress BV) were mixed and incubated at 37° C. for 30 minutes. Then, 25%glycerol containing bromophenol blue was added thereto at a finalglycerol concentration of 5%. Labeled ARC1172(wt) bound to vWF A1 domainwas separated from labeled ARC1172 in a free form by 10% non-denaturingpolyacrylamide gel electrophoresis. The gel was dried and visualizedusing Bio Image Analyzer FLA-7000, and the radioactivity was measured.The gel shift rate, the inhibition rate, and the relative binding ratewere calculated in accordance with Example 2.

<Results>

The binding inhibition ratio of each aptamer variant labeled ARC1172(wt)is shown in FIG. 18. When the binding inhibition ratio of the controlunlabeled ARC1172(wt) was defined as 1.00, the binding inhibition ratios(relative binding rates) of the aptamer variants in which inverted dTwas added to the 3′ end and to which mini-hairpin DNA was added were1.12 (ARC1172(wt)-iT), 1.37 (ARC1172-F), 1.43 (ARC1172-F-iT), and 1.61(ARC1172-G). ARC1172-G in which the hairpin structure was substitutedwith a mini-hairpin structure and further mini-hairpin DNA was added tothe 3′ end was shown to have the most improved binding activity, ascompared with the control ARC1172. These results demonstrated thatbinding strength can be improved by adding DNA to a conventional DNAaptamer consisting of natural bases.

Example 9: Analysis on Stability of Aptamer for vWF Against NucleolyticEnzyme, Etc.

In this Example, a DNA aptamer consisting of natural bases to whichmini-hairpin DNA was added was examined for its stability againstnucleolytic enzymes contained in human serum. Also, this technique wascomparatively studied with the addition of inverted dT to the 3′ end,which is a conventional technique of stabilizing nucleic acids.

<Method>

Each DNA aptamer containing or not containing the mini-hairpin DNA(ARC11721(wt), ARC1172(wt)-iT, ARC1172-F, ARC1172-F-iT, and ARC1172-G,final concentration: 2 μM) was mixed with human serum at a concentrationof 96%, and this solution was incubated at 37° C. After 0 hours, 1 hour,6 hours, 24 hours, 48 hours, and 72 hours, 10 μL was sampled from themixed solution, and the degradation reaction was terminated by mixingwith 110 μL of 1×TBE and a 10 M Urea solution. Each sample thus reactedwas fractionated by 15% denaturing polyacrylamide gel electrophoresis.Then, the gel was stained with SYBR GOLD to detect a single-strandednucleic acid. The band pattern of a degradation product by thenucleolytic enzymes in the human serum was analyzed using Bio ImagerLAS-4000 (Fujifilm Corp). The amount of residual DNA was predicted fromthe density of a band corresponding to an undegraded aptamer.

<Results>

The results are shown in FIG. 19. The band corresponding to thefull-length product of the control DNA aptamer (ARC1172(wt)) wasdegraded to 38% after 24 hours in the presence of human serum. The handcorresponding to the full-length product was degraded to 19% after 72hours. As compared with ARC1172(wt), the DNA aptamer to which inverteddT was added in accordance with the conventional technique ofstabilizing nucleic acids (ARC1172(wt)-iT) had improved stabilityagainst nucleolytic enzymes in human serum. Nonetheless, the bandcorresponding to its full-length product was degraded to 47% after 24hours in the presence of human serum. The band corresponding to thefull-length product was degraded to 27% after 72 hours. On the otherhand, the DNA aptamer in which mini-hairpin DNA was added both to the 3′end and to the internal sequence (ARC1172-G) was confirmed, from itsband pattern, to still retain 70% and 42% of the whole amount after 24hours and after 72 hours, respectively, in the presence of human serum.This demonstrated that stability against nucleolytic enzymes, etc.,contained in human serum is remarkably enhanced by the addition ofmini-hairpin DNA. The addition of mini-hairpin DNA to the 3′ end wasshown to be more effective for improving stability against nucleolyticenzymes, etc., contained in human serum, than the addition of inverteddT to the 3′ end (conventional technique of stabilizing nucleic acids).

Example 10: Analysis on Thermal Stability of Aptamer for vWF

<Method>

The thermal stability of each DNA aptamer (ARC1172(wt), ARC1172(wt)-iT,ARC1172-F, ARC1172-F-iT, and ARC1172-G; final concentration: 2 μM) wasstudied by the measurement of a Tm value. Change the absorbance of theDNA aptamer caused by the elevation of temperature (0.5° C./min) wasmeasured using an ultraviolet and visible spectrophotometer UV-2450(Shimadzu Corp.). The melting temperature (Tm value) was calculated fromthe first derivation thereof.

<Results>

The results are shown in FIG. 20. The control DNA aptamer (ARC1172(wt))had a Tm value of 63.8° C. whereas the DNA aptamer in which inverted dTwas added to the 3′ end in accordance with the conventional technique ofstabilizing nucleic acids (ARC1172(wt)-iT) had a Tm value of 63.2° C.The Ds aptamer variants containing the structure had a Tm value of 64.3°C. (ARC1172-F), 64.4° C. (ARC1172-F-iT), and 61.9° C. (ARC1172-G). Theseresults demonstrated that the addition of mini-hairpin DNA to the DNAaptamer neither largely changes the Tm value nor largely reduces thethermal stability.

Example 11: Design and Preparation of Aptamer for VEGF

In order to examine the influence of, for example, the addition of amini-hairpin structure and GC pairs, on the characteristics of aVEGF-binding DNA aptamer, each anti-VEGF165 DNA aptamer variant shown inFIG. 21 was designed and prepared.

Aptamer 47 (SEQ ID NO: 17) is a previously reported VEGF-binding DNAaptamer found by the inventors. Aptamer 47A (SEQ ID NO: 18) was derivedfrom Aptamer 47 by the substitution of the artificial base Ds with thenatural base A. Aptamer 49 (SEQ ID NO: 19) was a variant derived fromAptamer 47 by the elongation of the terminal stem structure by one G-Cbase pair. Aptamer 58mh (SEQ ID NO: 20) was derived from Aptamer 49 bythe further introduction of a mini-hairpin structure (short DNA fragmentconsisting of the sequence CGCGAAGCG (SEQ ID NO: 1)) to the 3′ end.Aptamer 49GC (SEQ ID NO: 21) and Aptamer 58GCmh (SEQ ID NO: 22) werederived from Aptamer 49 and Aptamer 58 mh by the substitution of A-Tbase pairs at two sites in the stem structure with G-C base pairs,respectively.

Each variant was chemically synthesized. In the same way as in Example1, each nucleic acid fragment was then purified by polyacrylamideelectrophoresis, heated at 95° C. in a phosphate buffer solution (pH7.4), and then slowly cooled to room temperature for reconstruction. Theresulting DNA aptamer was then used in Examples below.

Example 12: Analysis on Binding Activity of Aptamer for VEGF AgainstTarget Protein

<Method>

In order to compare binding activity against human-derived VEGF165protein among various aptamers prepared in Example 11, binding activityanalysis was conducted by the competition experiment. This analysis wasconducted at a scale of 20 μL. In a phosphate buffer solution (pH 7.4)containing 0.005% Nonidet P40, Aptamer 47 labeled with [γ-³²P]ATP (finalconcentration: 100 nM), each unlabeled variant (final concentration: 100nM), and VEGF165 protein (PeproTech, Inc., final concentration: 100 nM)were mixed and incubated at 37° C. for 30 minutes. Then, 5 μl of 25%glycerol was added thereto. Immediately thereafter, labeled Aptamer 47bound to VEGF165 protein was separated from free aptamer 47 by 10%non-denaturing polyacrylamide gel electrophoresis. The gel was dried andvisualized using Bio Image Analyzer, and the radioactivity was measured.The gel shift rate, the inhibition rate, and the relative binding ratewere calculated in accordance with Example 2.

<Results>

The results are shown in FIG. 22. As shown in FIG. 22, the aptamerderived from Aptamer 47 by the substitution with A (Aptamer 47A)exhibited no inhibition of binding, whereas Aptamer 49 derived fromAptamer 47 by the elongation of the stem region by one base pair had arelative binding rate of 1.53, demonstrating that its binding strengthwas significantly improved. Aptamer 49GC derived from Aptamer 49 by thesubstitution of A-T base pairs at two sites in the stem region with G-Cbase pair had a relative binding rate of 1.50. On the other hand,Aptamer 58mh and Aptamer 58GCmh containing the mini-hairpin had arelative binding rate of 1.58 and 1.59, respectively, and were thereforeconfirmed to have improved binding strength compared with Aptamer 47, aswith Aptamer 49.

Example 13: Analysis on Stability of Aptamer for VEGF in Human Serum

<Method>

In this Example, each DNA aptamer variant prepared in Example 11 wasused and examined for its stability in human serum.

Each DNA aptamer containing the mini-hairpin structure (Aptamer 58mh andAptamer 58GCmh) or each aptamer not containing this structure (Aptamer47, Aptamer 49, and Aptamer 49GC) (final concentration: 2 μM) was mixedwith human serum (Millipore Corp.) at a final concentration of 96%, andthis solution was incubated at 37° C., After 0 hours, 1 hour, 6 hours,24 hours, 48 hours, and 72 hours, 10 μL was sampled from the mixedsolution, and the degradation reaction was terminated by mixing with 110μL of 10 M urea/1×TBE. Each sample thus reacted was fractionated by 15%denaturing polyacrylamide gel electrophoresis containing 7 M urea. Then,the gel was stained with SYBR GOLD to detect a DNA fragment. The bandpattern of the DNA was analyzed using Bio Imager LAS-4000 (FujifilmCorp.). The amount of residual DNA was predicted from the density of aband corresponding to an degraded aptamer.

<Results>

The results are shown in FIG. 23. The band corresponding to thefull-length product of each aptamer not containing the mini-hairpinstructure (Aptamer 47, Aptamer 49, and Aptamer 49GC) was already 50% orless of the whole amount after 24 hours, whereas the band correspondingto the full-length product of each aptamer containing the mini-hairpinstructure (Aptamer 58mh and Aptamer 58GCmh) retained 50% or more of thewhole amount even after 72 hours. These results demonstrated that themini-hairpin structure allows the aptamer to be stabilized againstdegradation by nucleolytic enzymes in human serum.

Example 14: Analysis on Thermal Stability of Aptamer for VEGF inSolution

<Method>

The thermal stability of each variant (Aptamer 47, Aptamer 47A, Aptamer49, Aptamer 58mh, Aptamer 49GC, and Aptamer 58GCmh; final concentration:2 μM in a phosphate buffer solution, pH 7.4) was studied by themeasurement of a Tm value. Change in the absorbance of the DNA aptamercaused by the elevation of temperature (0.5° C./min) was measured usingan ultraviolet and visible spectrophotometer UV-2450 (Shimadzu Corp.).The melting temperature (Tm value) was calculated from the firstderivation thereof.

<Results>

The results are shown in FIG. 24. The control DNA aptamer (Aptamer 47)had a Tm value of 69.5° C., whereas each variant without thesubstitution with G-C base pairs had a Tm value of 60.0° C. (Aptamer47A), 67.7° C. (Aptamer 49), and 65.6° C. (Aptamer 58mh). Thus, theaddition of the mini-hairpin structure and G-C base pairs did notlargely change the Tm value itself. As for Aptamer 49GC and Aptamer58GCmh substituted with G-C base pairs, two phases of melting curveswere obtained, and their Tm values were 54.6° C. and 78.7° C. forAptamer 49GC and 62.6° C. and 80.8° C. for Aptamer 58GCmh. In order tofurther specifically examine the influence of the elongation by the G-Cbase pair, the substitution, and the addition of the mini-hairpinstructure, melting curves up to 50° C. were enlarged and shown in thelower column of FIG. 24. It was confirmed that the change in absorbancewas suppressed by the elongation by the G-C base pair (Aptamer 49) andthe addition of the mini-hairpin structure (Aptamer 58mh) to stabilizethe structure, and the structure was further stabilized by thesubstitution with G-C base pairs (Aptamer 49GC and Aptamer 58GCmh).

All of the publications, patents, and patent applications areincorporated herein by reference.

The invention claimed is:
 1. A method for producing a DNA aptamer beingstabilized and/or having enhanced capacity to bind to a target molecule,comprising the steps of: designing a base sequence of a DNA aptamer inaccordance with a method for designing a base sequence that enhances thecapacity of an existing DNA aptamer to bind to a target molecule and/orstabilizes the aptamer; and producing a DNA aptamer on the basis of thedesigned base sequence, wherein the method for designing a base sequencecomprises substituting a hairpin structure consisting of one pair ofstem structure and loop structure of the DNA aptamer comprising at leastone stem structure and at least one loop structure with a mini-hairpinstructure, wherein the stem structure is formed by base pairing of 2 ormore consecutive bases constituting one strand with bases constitutinganother strand, wherein the mini-hairpin structure comprises: nucleicacid regions (A) to (C) below sequentially ligated from the 5′ endtoward the 3′ end: (A) a first nucleic acid region comprising 2 to 5arbitrary nucleotides; (B) a second nucleic acid region comprising a“gna” or “gnna” base sequence wherein each “n” independently representseither “g”, “t”, “a”, or “c”, a base analogue, or a modified base; and(C) a third nucleic acid region comprising a base sequence complementaryto the first nucleic acid region, wherein the first nucleic acid regionand the third nucleic acid region form a stem portion by base pairingwith each other and the second nucleic acid region forms a loop portion.2. The method according to claim 1, wherein the DNA aptamer comprises atleast one base analogue and/or modified base.
 3. The method according toclaim 2, wherein the base analogue is7-(2-thienyl)-3H-imidazo[4,5-b]pyridin-3-yl.
 4. The method according toclaim 1, comprising increasing one or more GC pairs in a stem structureother than that constituting the mini-hairpin.
 5. The method accordingto claim 1, wherein the DNA aptamer comprises one of the at least onestem structure at the 5′ and/or 3′ ends thereof, wherein the methodcomprises increasing one to five GC pairs in the 5′ and/or 3′ ends ofthe DNA aptamer.
 6. The method according to claim 1, comprising adding afurther mini-hairpin structure as defined in claim 1 to one end of theDNA aptamer.
 7. The method according to claim 1, wherein the differencein nucleotide length between the hairpin structure to be substituted andthe mini-hairpin structure after the substitution is 3, 2, 1, or 0.